DESCRIPTION: The proposed research project will investigate the cellular and molecular basis for segment pattern formation in the Helobdella leech embryo. The leach was chosen as a model system because the close phylogenetic relationship to insects offers a good opportunity for comparative analysis to the well studied early developmental genes in Drosophila. Dr. Savage notes that although annelids and arthropods are segmented and thought to diverge from a common ancestor, the embryonic stages are dissimilar. This comparative study will examine the degree of conservation of developmental mechanisms. Dr. savage proposes to study the expression and translational regulation of the Helobdella Lzf2 (Leech Zinc Finger) gene, which is an orthologue of the Drosophila gap gene hunchback (hb). Lzf2 is a gene Dr. Savage identified in the leech as a postdoctoral student. The proposed experiments are designed to characterize the translation and distribution of the Lzf2 protein, to determine if the Lzf2 distribution is regulated by translational inhibition and to characterize Lzf2 function by antisense technology. Dr. Savage is currently testing the specificity of antisera generated against a Lzf2-GST fusion protein by immunoprecipitation of the leech protein from the bacterial protein, by Western blotting and most importantly by immunostaining leech embryos. The immunostaining will follow well established procedures, with special attention paid to teloblasts (stem cells leading to segmental mesoderm and ectoderm), post-mitotic neurons and staining along the body axis. The different Lzf2 expressing cells will be followed by cell lineage tracer injections with rhodamine-dextran. The same embryos will be stained with fluorescein-conjugated secondary antibody to determine if the immunopositive cell derives from the injected cell. The second aim is to demonstrate that the NRE-like sequences (Nanos Related Element) found in the Lzf2 3' UTR are capable of translational inhibition. Dr. Savage proposes to tag the NRE-like sequences to the 3' UTR of two reporter genes (B-galactosidase and green fluorescent protein) and inject the mRNAs to assay for presence of reporter gene activity in the injected stem cell and its progeny blast cells. If the Nanos protein is present in these cells, then it might bind, with other proteins to the NRE-like sequences and translationally repress the reporter protein. These results would help demonstrate that the fly hb gene and the leech Lzf2 orthologue are under a conserved regulation. The third aim is the misregulation of Lzf2 during embryogenesis to assess its developmental role. The intent is to reduce Lzf2 protein expression by injecting multiple 20-25 base length oligonucleotides that are complementary to different regions of the Lzf2 coding sequence to deplete mRNA, or to inject full length antisense mRNA. Reduced levels of Lzf2 protein will be assayed by immunocytochemistry and Western blot analysis. A tracer will be co-injected into the uncleaved egg and tracked through early cleavage divisions, teloblast formation and through segment patterning of late stage embryos. Control experiments will be initiated as needed. The last experiment will assess the effect of over expression and ectopic expression of lzf2 by injecting capped mRNA lacking the NRE-like sequences into teloblasts, along with a tracer and examine developmental abnormalities, specifically looking for changes in segment identity.